The two major cellular components of the vasculature are the endothelial and smooth muscle cells. The endothelial cells form the lining of the inner surface of all blood vessels, and constitute a nonthrombogenic interface between blood and tissue. In addition, endothelial cells are an important component for the development of new capillaries and blood vessels. Thus, endothelial cells proliferate during the angiogenesis, or neovascularization, associated with tumor growth and metastasis, as well as a variety of non-neoplastic diseases or disorders.
Various naturally occurring polypeptides reportedly induce the proliferation of endothelial cells. Among those polypeptides are the basic and acidic fibroblast growth factors (FGF), Burgess and Maciag, Annual Rev. Biochem., 58:575 (1989), platelet-derived endothelial cell growth factor (PD-ECGF), Ishikawa, et al., Nature, 338:557 (1989), and vascular endothelial growth factor (VEGF), Leung, et al., Science 246:1306 (1989); Ferrara & Henzel, Biochem. Biophys. Res. Commun. 161:851 (1989); Tischer, et al., Biochem. Biophys. Res. Commun. 165:1198 (1989); Ferrara, et al., PCT Pat. Pub. No. WO 90/13649 (published Nov. 15, 1990).
VEGF was first identified in media conditioned by bovine pituitary follicular or folliculostellate cells. Biochemical analyses indicate that bovine VEGF is a dimeric protein with an apparent molecular mass of approximately 45,000 Daltons, and with an apparent mitogenic specificity for vascular endothelial cells. DNA encoding bovine VEGF was isolated by screening a cDNA library prepared from such cells, using oligonucleotides based on the amino-terminal amino acid sequence of the protein as hybridization probes.
Human VEGF was obtained by first screening a cDNA library prepared from human cells, using bovine VEGF cDNA as a hybridization probe. One cDNA identified thereby encodes a 165-amino acid protein having greater than 95% homology to bovine VEGF, which protein is referred to as human VEGF (hVEGF). The mitogenic activity of human VEGF was confirmed by expressing the human VEGF cDNA in mammalian host cells. Media conditioned by cells transfected with the human VEGF cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not. See, Leung, et al., Science 246:1306 (1989).
Several additional cDNAs were identified in human cDNA libraries that encode 121-, 189-, and 206-amino acid isoforms of hVEGF (also collectively referred to as hVEGF-related proteins). The 121-amino acid protein differs from hVEGF by virtue of the deletion of the 44 amino acids between residues 116 and 159 in hVEGF. The 189-amino acid protein differs from hVEGF by virtue of the insertion of 24 amino acids at residue 116 in hVEGF, and apparently is identical to human vascular permeability factor (hVPF). The 206-amino acid protein differs from hVEGF by virtue of an insertion of 41 amino acids at residue 116 in hVEGF. Houck, et al., Mol. Endocrin. 5:1806 (1991); Ferrara, et al., J. Cell. Biochem. 47:211 (1991); Ferrara, et al., Endocrine Reviews 13:18 (1992); Keck, et al., Science 246:1309 (1989); Connolly, et al., J. Biol. Chem. 264:20017 (1989); Keck, et al., EPO Pat. Pub. No. 0 370 989 (published May 30, 1990).
Receptors for VEGF have been described in the literature. Two such receptors, flt-1 and flk-1, have been found to mediate VEGF effects [DeVries et al., Science 255:989 (1992); Shibuya et al., Oncogene 5:519 (1990); Matthews et al., Proc. Natl. Acad. Sci. 88:9026 (1991); Terman et al., Oncogene 6:1677 (1991); Terman et al., Biochem. Biophys. Res. Comm. 187:1579 (1992); Neufeld et al., Prog. Growth Factor Res. 5:89-97 (1994); Waltenberger et al., J. Biol. Chem. 269:26988 (1994); Quinn et al., Proc. Natl. Acad. Sci. 90:7533 (1993)], but their regulation and mechanisms are not yet fully understood. Lennmyr et al., J. Neuropathology and Exp. Neurology 57:874-882 (1998). Both the flt-1 and flk-1 receptors are membrane-spanning receptors and belong to the class III tyrosine kinase receptor family. Barleon et al., J. Cell Biochem. 54:56 (1994); Neufeld et al., supra.
VEGF not only stimulates vascular endothelial cell proliferation, but also induces angiogenesis. Angiogenesis, which involves the formation of new blood vessels from preexisting endothelium, is an important component of a variety of diseases and disorders including tumor growth and metastasis, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, age-related macular degeneration, hemangiomas, immune rejection of transplanted corneal tissue and other tissues, and chronic inflammation.
In the case of tumor growth, angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment to the growing solid tumor. Folkman, et al., Nature 339:58 (1989). Angiogenesis also allows tumors to be in contact with the vascular bed of the host which may provide a route for metastasis of the tumor cells. Evidence for the role of angiogenesis in tumor metastasis is provided, for example, by studies showing a correlation between the number and density of microvessels in histologic sections of invasive human breast carcinoma and actual presence of distant metastases. Weidner, et al., New Engl. J. Med. 324:1 (1991).
VEGF has also been reported to be involved in endothelial and vascular permeability. See, Ferrara et al., Endocrine Reviews 18:4-25 (1997); Dobrogowska et al., J. Neurocytology 27:163 (1998). Although not fully understood, VEGF is believed to increase endothelial cell leakage in skin, retina, and tumor tissues. Collins et al., Brit. J. Pharmacology 109:195 (1993); Connolly et al., J. Clin. Invest. 84:1470 (1989); Shweiki et al., Nature 359:843 (1992); Monacci et al., Am. J. Physiol. 264:C995 (1993); Stone et al., J. Neurosci. 15:4738 (1995); Detmar et al., J. Invest. Dermatol. 108:263 (1997); Weindel et al., Neurosurgery 35:437 (1994). The potential effects and role of VEGF (and its receptors, particularly, the flt-1 receptor), on endothelial cell and blood-brain barrier permeability have also been examined. See, e.g., Rosenstein et al., Proc. Natl. Acad. Sci. 95:7086 (1998); Dobrogowska, supra; Kovacs et al., Stroke 27:1865 (1996). Relatively diffuse VEGF mRNA expression has been observed in adult rat brain but at somewhat low abundance. Monacci et al., Am. J. Physiol. 146:368-378 (1993). However, reduced oxygen tension has been shown to trigger VEGF expression [Dor and Keshet, Trends in Cardiovascular Med., 7:289-294 (1998)] and enhanced levels of VEGF, flt-1, and flk-1 have been shown to occur in the rat brain following the induction of focal cerebral ischemia. Hayashi et al., Stroke 28:2039 (1997); Kovacs et al., supra; Lennmyr et al., J. Neuropathology and Experimental Neurology, 57:874 (1998). The role of VEGF in the pathogenesis of stroke and BBB breakdown has been unclear with contradictory experimental observations cited in the literature. For example, Nag et al., J. Neuropathology and Experimental Neurology 56:912 (1997), in their cortical cold-injury rat model, demonstrated the presence of mural VEGF in permeable pial vessels and arterioles within the damaged tissue and, from this observation, it was inferred that VEGF is one of several factors that may mediate BBB breakdown and edema formation. On the other hand, in Hayashi et al., J. Cerebral Blood Flow and Metabolism, 18:887 (1998), it is reported that VEGF itself, when applied topically on the surface of a reperfused rat brain after transient cerebral artery occlusion, reduced ischemic brain damage, infarct volume and edema formation.